Homocystinuria due to cystathionine synthase deficiency. Studies of nitrogen balance and sulfur excretion.

J R Poole, … , E B Conerly, W A Edwards

J Clin Invest. 1975;55(5):1033-1048. https://doi.org/10.1172/JCI108004.

Research Article

Apparent nitrogen balances and urinary sulfur excretions were determined for normal subjects, seven cystathionine synthase-deficient patients, and a single cystathioninuric patient on semisynthetic diets containing low-adequate amounts of and very low amounts of methionine and very low amounts (12 mg daily, or less) of cystine. The amounts of supplemental cystine required to prevent abnormally high nitrogen or sulfur losses were determined. The five cystathionine synthase-deficient patients who had low residual activities of this enzyme detected in fibroblast and/or liver extracts did not lose more nitrogen or sulfur on diets virtually devoid of cystine than did the normal subjects. These results suggest that the widely expressed opinion that cystine is an essential for cystathionine syntase- deficient patients requires modification. Residual enzyme activity of only a few percent of normal may obviate such a cystine requirement. These results are compatible with, and lend support to, the working hypothesis which states that the pyridoxine response in cystathionine synthase-deficient patients is mediated by an increase in the residual activity of the affected enzyme.

Find the latest version: https://jci.me/108004/pdf Homocystinuria due to Cystathionine Synthase Deficiency

STUDIES OF NITROGEN BALANCE AND SULFUR EXCRETION

JEFFRmY R. PooLE, S. HARVEY MUDD, E. BRINSON CONELY, and WILLIAM A. EDWARDS From the Digestive and Hereditary Diseases Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, and the Laboratory of General and Comparative Biochemistry, National Institute of Mental Health, Bethesda, Maryland 20014

A B S T R A C T Apparent nitrogen balances and urinary (reaction 1). Cystathionine is cleaved by y-cystathi- sulfur excretions were determined for normal subjects, onase (EC 4.4.1.1) to form and a-ketobutyrate seven cystathionine synthase-deficient patients, and a (reaction 2): single cystathioninuric patient on semisynthetic diets cystathionine containing low-adequate amounts of methionine and + synthase very low amounts (12 mg daily, or less) of cystine. The amounts of supplemental cystine required to prevent cystathionine + H20 (1) y-cystathionase abnormally high nitrogen or sulfur losses were deter- H20 mined. The five cystathionine synthase-deficient patients cystathionine + - who had low residual activities of this enzyme detected cysteine + a-ketobutyrate + NH3 (2) in fibroblast and/or liver extracts did not lose more serine -> nitrogen or sulfur on diets virtually devoid of cystine homocysteine + cysteine than did the normal subjects. These results suggest that +a-ketobutyrate + NH3 (1) + (2) the widely expressed opinion that cystine is an essential Humans have been discovered with genetically de- amino acid for cystathionine synthase-deficient patients termined deficient activity of either cystathionine syn- requires modification. Residual enzyme activity of only thase (1, 2) or y-cystathionase (3-5), the hallmark a few percent of normal may obviate such a cystine re- excretory abnormalities of which are, respectively, quirement. These results are compatible with, and lend homocystinuria and . support to, the working hypothesis which states that the Early in the study of homocystinuria, it was pointed pyridoxine response in cystathionine synthase-deficient out that a block in the transsulfuration pathway would patients is mediated by an increase in the residual ac- be expected to decrease the ability to synthesize cysteine tivity of the affected enzyme. (1, 6). Normal humans do not require exogenous cyst (e) ine (7, 8). In contrast, it seemed possible that INTRODUCTION patients with sufficiently severe cystathionine synthase deficiency would require an exogenous supply of this The transsulfuration pathway in mammals provides a amino acid. For these patients, cyst (e) ine would have route whereby the sulfur of methionine becomes the become an essential amino acid (1, 6). This theoretical sulfur of cyst (e) ine. Methionine is demethylated via expectation was apparently confirmed when Brenton, two intermediates to form homocysteine, which is com- Cusworth, Dent, and Jones carried out nitrogen bal- bined with serine by the action of cystathionine syn- ance studies on a single homocystinuric patient. This thase (EC 4.2.1.22) to yield the thioether, cystathionine patient went into negative nitrogen balance when daily (HOOC (NH2) CHCH2CH2SCH2CH(NH2)COOH) cystine intake was decreased to 85 mg. Nitrogen bal- Received for publication 17 April 1974 and in revised form ance was restored when the cystine intake was increased 27 December 1974. to 1,085 mg (9). In subsequent reviews and texts it has The Journal of Clinical Investigation Volume 55 May 1975.1033-1048 1033 TABLE I Patients

Admission Responsive to Patient Sex Age weight Demonstration of enzyme deficiency pyridoxine yr kg Cystathionine synthase deficient Wi. Cr. M 15 79.3 Fibroblast assay (patient 16, reference 18) Yes* Bu. Ha. M 8 29.3 Fibroblast assay (patient 32, reference 18) Not Jo. Ho. F 31 69.5 Liver, fibroblast assays (patient 31, references 18, 19) Yes (19) Ja. Is. F 13 62.4 Fibroblast assay (patient 1, reference 18) Yes§ Ro. Kr. F 20 63.9 Fibroblast assay (patient 6, reference 18) Yes§ Th. Kr. M 31 79.5 Fibroblast assay (patient 8, reference 18) Yes (19) 01. Ma. F 19 55.9 Fibroblast assay (patient 34, reference 18) Noll Ca. Me. F 20 64.5 Fibroblast assay¶ Yes§ Fr. Mi. M 12 54.5 Fibroblast assay¶ No** Ba. Re. F 31 67.6 Fibroblast assay (patient 35, reference 18) No§ Vi. Sw. F 17 62.0 Fibroblast assay¶ Nott Cystathioninuric Ge. Po. M 21 74.9 Indirect evidence only§§ Yes * Personal communication from Dr. A. Sass-Kortasak. Patient previously reported (20). 1 Personal communication from Dr. N. Holtzmann. § Personal communication from Dr. V. McKusick. 11 Patient's urinary and plasma homocystine and methionine values remained essentially unchanged despite the ad- ministration of 500 mg pyridoxine - HCl daily for 11 days. Serum folate was normal. ¶ Previously unpublished specific activities of cystathionine synthase, assayed in fibroblast extracts without addition of pyridoxal phosphate, or in the presence of pyridoxal phosphate added at final concentrations of 0.1, 1.0, and 5.0 mM were as follows: Ca. Me., 0.46, 0.64, 0.83, 0.73; Fr. Mi., 0.59, 0.70, 0.96, 0.65 (mean of two assays); Vi. Sw. 0, 0, 0, 0 (i.e., <0.10). Activities are in nmol cystathionine/mg protein/135 min; control mean, 31.7; range, 3.7-60.0 (18). ** Personal communication from Dr. G. Morrow, III. Morrow's observation was confirmed during the present studies. Hypermethioninemia, homocystinemia, hypocystinemia, and homocystinuria all remained relatively constant during 10 days of treatment with a daily oral dose of 500 mg pyridoxine- HCl. tt After taking 3 mg folic acid and 300 mg pyridoxine * HCl daily for several months, the patient had elevated urinary homocystine and methionine levels. 1 mo after decreasing the folic acid to 2 mg twice weekly and discontinuing the pyridoxine, no significant change in urinary homocystine or methionine was noted. §§ Cystathionine was elevated in plasma and urine. Patient was free of nongenetic conditions known to cause cysta- thioninuria (21). 11 The classification of Ge. Po. as a responder to B6 is based upon the response of his cystathioninuric brother (21). been widely and generally stated that cystine is an will be reported, we found that under the conditions essential amino acid for cystathionine synthase-deficient employed those cystathionine synthase-deficient patients patients (2, 10-17). with low, but detectable, residual enzyme activities had Recent indications of genetic heterogeneity in cysta- no greater need for exogenous cystine than did normal thionine synthase-deficient patients (18), in conjunc- volunteer subjects. tion with an increasing realization of the possible func- tional significance of small residual activities of cysta- METHODS thionine synthase in deficient patients (19), led us to Subjects. The control subj ects were normal volunteers question whether the generally accepted opinion of the aged 18-24 yr, in good health as indicated by medical his- essential nature of cyst (e) ine for such patients were tories, physical examinations, and laboratory tests, including not an As more in urinary amino acid analyses. oversimplification. explained fully Patients. Information regarding the patients is summa- the Discussion, it seemed possible that patients with as rized in Table I. Presently, certain vitamin Bs-responsive little as 1-2% of the normal activity of cystathionine cystathionine synthase-deficient patients are maintained, as a synthase might not require cyst(e)ine. To test this therapeutic measure, on large supplemental doses of pyri- hypothesis, we determined the amount of cystine re- doxine (16, 17, 22). To study such patients in "basal" to states, their vitamin B6 intakes were limited to that con- quired maintain short-term nitrogen balance in a tained in their diets for a minimum of 1 mo before the series of cystathionine synthase-deficient patients. As beginning of the present studies. This was sufficient time 1034 J. R. Poole, S. H. Mudd, E. B. Conerly, and W. A. Edwards for the urinary homocystine excretions of responsive patients tity of hydrochloride salt); L-methionine, 0.63; L-phenylala- previously under treatment with pyridoxine to return to nine, 1.16; L-, 1.00; L-, 0.33; L-, pretreatment levels (19) and for urinary 4-pyridoxic acid 0.86; L-, 1.49. For patient Fr. Mi. an additional 300 excretions to return to normal (see Results). The cysta- mg of L- and 170 mg of L-methionine were added to thioninuric patient had not recently received pyridoxine meet the published amino acid requirements for his age treatment. group (24). Otherwise, methionine and cystine were added Experimental plan. During the present work, several as specified in the individual studies. was added to studies were performed, each requiring a different diet. (a) bring the total supplemental amino acid nitrogen to 10.0 g. When nitrogen balance and sulfur excretion studies were Thus, total nitrogen content of the experimental diet (food performed concomitantly, each subject received a constant nitrogen plus supplemental amino acid nitrogen) ranged natural diet (the "equilibration diet") until nitrogen bal- from 10.4 to 11.2 g/day, depending on each subject's weight ance was attained (approximately 6-15 days), after which and caloric requirements. he was placed on a semisynthetic diet (the "experimental Vitamin and mineral supplements. While on the experi- diet") containing approximately the same amount of nitro- mental diet all subjects received one 0.5-mg pyridoxine-HCl gen as the equilibration diet. For a period of 0-3 days after capsule daily and one especially formulated combination switching to the experimental diet, adjustments were made vitamin and mineral capsule which supplied 10,000 U vita- in caloric intake and the other dietary components until min A, 400 U vitamin D, 5 mg thiamine mononitrate, 5 the subject's weight remained stable and the diet was accept- mg riboflavin, 25 mg niacinamide, 2 Aig cyanocobalamin, 5 able to him. Thereafter, the experimental diet became con- mg calcium pantothenate, 100 mg ascorbic acid, 10 mg fer- stant, and was divided into 6-day dietary periods, during rous sulfate, 1 mg copper sulfate, 150 lig calcium iodate, 1 each of which a specified amount of L-cystine was added mg manganese sulfate, 5 mg magnesium oxide, and 1.5 mg to the diet. During all studies the subjects were inpatients zinc sulfate. During initial studies, five subjects (Wi. Cr., at the Clinical Center of the National Institutes of Health. Ba. Ha., Ba. Re., Ka. Ro., and An. To.) did not receive They were instructed to maintain reasonably constant levels these two capsules, but instead one commercial multivitamin of activity, avoiding strenuous exercise. 24-h urine collec- tablet (Dayalets-M, Abbott Laboratories, North Chicago, tions were made under toluene and were refrigerated until Ill.) daily. The contents of this tablet differed from those volumes were measured and samples frozen. Stool collec- of the especially formulated combination vitamin and mineral tions were pooled for each 72-h period. Fasting blood speci- capsule only in that the commercial tablet provided a total mens were obtained during the latter half of each dietary of 2.0 mg of pyridoxine HCl and approximately twice the period. (b) For sulfur partition studies, subjects received amount of each of the minerals. Throughout all of the stud- constant natural diets calculated to provide half of the ies, each subject received 2 mg of folic acid twice weekly. dietary sulfur in the form of methionine and half in the Medications. Patients Jo. Ho., Ca. Me., and Ba. Re. each form of cystine. These diets provided approximately 0.14 necessarily received certain medication(s) during the present mmol of methionine and 0.07 mmol of cystine (i.e., 0.14 studies. Patient Jo. Ho. received 60 mg isoxsuprine *HCl mg-atom of cystine sulfur)/kg body wt. daily throughout all of the studies. Patient Ca. Me. received Diets. All of the diets were prepared and served under 30 mg phenobarbital twice daily and 100 mg diphenylhy- the supervision of a dietician trained in the techniques of dantoin twice daily throughout the studies. Patient Ba. Re. metabolic balance studies. The equilibration diets provided received 75 mg of amitriptyline *HCl and 50 mg hydro- between 10.0 and 11.2 g of nitrogen (as indicated by calcu- chlorthiazide daily throughout her first two studies. Urinary lation and analyses), 1,175-1,563 mg of methionine, and sulfur partition (Table VI) was measured during the first 912-1,197 mg of cystine. These dietary amino acid intakes study, and nitrogen balances on 109, 309, and 509 mg cystine were calculated from published tables (23). (Table IV) were determined during the second. During The semisynthetic experimental diet consisted of foods her second study she was also given 300 mg and subse- and beverages low in methionine and cystine, including cook- quently 400 mg of sulfinpyrazone daily. During her third ies, muffins, and pancakes prepared from Cellu Low Pro- study (nitrogen balance on 8 and 1,008 mg cystine [Table tein Baking Mix, Cellu Pasta Imitation Macaroni (both IV]) she was no longer on any of the above medications, Cellu products from Chicago Dietetic Supply House, Inc., and she received 50 mg spironolactone and 400 mg of dipy- La Grange, Ill.), butter oil, jelly, air mints, sour balls, vine- ridamole daily. gar, oil, various vegetables, fruits, and fruit juices, car- Methionine loads. For methionine loading studies, the bonated beverages, coffee, tea, and sugar. Controlyte (The patient was placed on the constant natural diet used for Doyle Pharmaceutical Company, Minneapolis, Minn.), a sulfur partition studies. After the total sulfur output had high calorie dietary supplement with trace protein content remained relatively constant for several days, L-methionine (1.4 mg methionine and 0.7 mg cystine/100 g) was used in was given in gelatin capsules, each containing 0.25 g of L- the form of a fruit-flavored beverage as a source of calories methionine. The subject was fasted from 10 p.m. the pre- and as a vehicle for the supplemental amino acids. Together, vious night, and the amino acid was administered at 8 a.m. these materials provided 0.4-1.2 g of nitrogen (calculation in a dose of 0.5 mmol/kg body wt. Breakfast was fed at and analysis), approximately 14-55 mg of methionine (cal- 9 a.m. 24-h urines were collected before and after the culation), and 3-12 mg of cystine (calculation). To these methionine load. materials were added supplemental amino acids as shown Analytical methods. Nitrogen analyses were performed below and in the individual studies. by use of a micro-Kjeldahl method (25). Values obtained Amino acids. The eight essential amino acids plus ar- for fecal nitrogen excretions over 72 h were divided by 3 ginine, , and tyrosine were added to the experi- to give mean daily fecal nitrogen outputs. Urinary creatin- mental diet in their pure L-forms in the approximate quanti- ines were determined by the method of Chasson, Grady, and ties found in 20 g of whole egg protein. The amounts added Stanley (26). Urinary and plasma amino acids were de- were (in grams per day): L-, 1.31 (as equivalent termined with an automatic amino acid analyzer with the quantity of hydrochloride salt); L-histidine, 0.48; L-isoleu- buffer system of Spackman, Stein, and Moore (27). 4- cine, 1.33; L-, 1.76; L-lysine, 1.28 (as equivalent quan- Pyridoxic acid concentrations in the urine were determined N Balance and S Excretion in Cystathionine Synthase Deficiency 1035 by the microprocedure of Woodring, Fisher, and Storvick distilled H20 to a 50-ml beaker. A clear blue solution (28). Serum folate concentrations were determined by the should result from this procedure. Subsequent steps were procedure of Harper (29). the same as those used for the 10-ml acidified aliquot ana- Differential analyses of urinary inorganic sulfate, total lyzed for inorganic sulfate except that the precipitate was sulfate (i.e., inorganic sulfate + "ethereal" sulfate), and washed once with 2 ml cold 50% acetone and then with 2 total sulfur were performed according to the general pro- ml and 5 ml cold 95% acetone. cedure of Rosenheim and Drummond, as specified in Hawk, Further modifications were required for samples very low Oser, and Summerson (30). A detailed description is given, in sulfur because of low dietary intake. Initial samples of since the methods we are currently using involve a number approximately 1% of a daily urinary volume were diluted of modifications of the original method. to 21 ml. 10 ml of the diluted specimen was treated for For phosphate removal, up to 12.5 ml of urine was diluted phosphate removal with half the reagent quantities previ- with water to a volume of approximately 25 ml in a 50-ml ously described and ending with a volume of 25 ml. Appro- volumetric flask, and the solution made basic with NH&OH priate aliquots, usually 7.5 ml, were then treated as usual (approximately 5 N) with phenolphthalein as indicator. except that precipitates were titrated with 0.005 N NaOH. After addition of 5 ml 5% NH4Cl and an aqueous slurry Precipitates containing less than 5 ,umol or more than 25 containing 0.75 g MgCOa (Baker reagent: 4MgCOasMg jsmol sulfate often gave unsatisfactory results, and sample (OH)5 nH2O) in 5 ml, the preparation was diluted to sizes were adjusted accordingly. It was also found that total volume and inverted five times. The mixture was inverted sulfur results tended to be spuriously low if the starting at 3-min intervals for 15 min, allowed to stand for 15 min, sample exceeded 1-2% of the total daily urine volume. mixed again, and centrifuged. Aliquots of the supernatant Nonsulfate sulfur was calculated as total sulfur minus total fluid were taken for analyses. sulfate. Since this value and that for ethereal sulfate (total For analysis of inorganic sulfate, an aliquot (usually 5 sulfate minus inorganic sulfate) usually depend upon rela- ml) of the phosphate-free supernatant fluid was brought to tively small differences between two larger experimentally a total volume of 10 ml in a 50-ml beaker and acidified determined quantities, it was important to perform the with HC1 to bromphenol blue. To precipitate sulfate ions, three determinations (inorganic sulfate, total sulfate, total the sample was chilled for 15 min at 4VC, and mixed with sulfur) for a given specimen in parallel during a single 2 m cold benzidine reagent (5 g benzidine dihydrochloride analytical run. All determinations were done in duplicate, and 50 ml 1 N HC1 in a total volume of 250 ml, made and the mean result is reported. Usually four urine samples fresh each week). After 5 min at 4VC, 5 ml 95% acetone could be handled in this way during a single day. Deter- was added, and the mixture allowed to stand an additional minations were repeated if disagreement between duplicates 15 min at 40C. Precipitate was isolated by filtration through was more than 10%. glass fiber filter paper in Gooch crucibles and washed twice Fecal specimens were assayed for total sulfur only by a with 2-ml portions of cold 95%o acetone and once with a modification of the above method. After collection, samples 5-ml portion. The precipitate, together with the filter paper, were homogenized with approximately 2 weights H20 and was transferred to a 250 ml Erlenmeyer flask. Boiling water stored frozen. Aliquots of 3 g total weight were mixed with was used to wash the crucible to ensure complete transfer further H20 to a total volume of 10 ml. 0.5 ml Benedict's and to suspend the filter paper and precipitate. The pre- reagent was added, and the suspension was dried in an cipitate dissolved slowly during the subsequent titration. evaporating dish over boiling H20 and heated for 3 h in a Titration was carried out with 0.01 N NaOH at approxi- muffle oven, attaining a temperature of 3000 C within an mately 100°C with constant mechanical stirring to a lasting hour and a final temperature of 650-700'C. After cooling, phenol-red end point. The normality of the NaOH was the residue was dissolved in 1 ml 2 N HCO and dried. The determined at each use by titration against commercial stan- cooled residue was reconstituted with 1 drop 2 N HCO and dard HC1. two 5-ml portions of I{O and transferred to a 25-ml volu- For analysis of total sulfate (inorganic and ethereal), metric flask. Phosphate removal, as previously described, an aliquot (5-10 ml) of the phosphate-free supernatant fluid was carried out by using the appearance of the intense blue was subjected to acid hydrolysis to convert ethereal sulfate color of the copper-ammonia complex ion as indicator, cor- to inorganic sulfate. The aliquot was mixed with 1 ml 1 N responding to a pH of 7.0-7.5. 10 ml of the phosphate-free HC1 in a porcelain evaporating dish and taken to dryness supernatant fluid was then assayed according to the usual over boiling water. (Unless otherwise specified, heating and procedure for inorganic sulfate. In 13 runs using this pro- drying were routinely done on a boiling water bath.) The cedure the mean recovery of an added mixture containing residue was transferred to a 50-ml beaker with two 5-ml equal amounts of methionine sulfur and cyst (e) ine sulfur portions of distilled water. No acidification of this sample was 56.8+3.3% (SE) (range, 34-74%). was required before the subsequent steps, which were the Growth of fibroblasts and sensitive assay of cystathionine same as those described above for the acidified aliquot synthase activity. These procedures were performed essen- analyzed for inorganic sulfate. tially as previously described (18). For analysis of total sulfur, an aliquot (5-10 ml) of the Treatment of the data. In calculating nitrogen balances, phosphate-free supernatant fluid was subjected to oxidation only those days were considered during which the patients to ensure that all sulfur would be in the form of inorganic were on the experimental diets and adjustments of caloric sulfate. The aliquot was taken to dryness in an evaporating intakes had been completed. From these, all days in which dish together with 0.25 ml Benedict's reagent (20% Cu urine collections were lost or known to be incomplete were [N01s,3HO, 5%o KClOs), reconstituted with 1 ml dis- eliminated, leading to the omission of five of a potential tilled H20 and 0.25 ml Benedict's reagent, and again taken total of 204 subject-days for the studies reported here. The to dryness. The residue was heated to redness with a Bun- urinary excretions for the remaining days for sen burner for 3 min. The carbon-free residue was re- each patient were then analyzed by the methods of Grubbs constituted with 1 ml 2 N HCI and taken to dryness. One to identify any statistical outliers (31). Excretions for these drop of 2 N HC1 was added to the evaporating dish, and outlying days (a total of 11) were considered suspect and the dry residue was transferred with two 5-ml portions of were eliminated from the subsequent analyses. Nitrogen bal- 1036 J. R. Poole, S. H. Mudd, E. B. Conerly, and W. A. Edwards ances were then obtained by subtracting the daily values TABLE 1 I for urinary and fecal nitrogen from the total nitrogen intake provided by food, vitamin and mineral capsules, supple- Urinary 4-Pyridoxic Acid Excretions mental amino acids, and medications. Excess urinary sulfur calculated as 4-Pyridoxic acid excretion Pyridoxine - HC was dietary sulfur supplement on intake minus urinary sulfur excretion. The shift from the Equilibration Experimental experimental equilibration diets to the experimental diets involved de- Subject diet diet diet* creases in total sulfur intakes (unlike nitrogen intakes, which remained relatively constant). Thus, sulfur excretions mg/day mg/day usually decreased during the first 2 days on the experi- Normal volunteers mental diets and assumed more or less constant values from Ba. Ha. 0.75 1.13 2.0 the 3rd day through the end of the period. For this reason, Ka. Ro. 0.80 1.25 2.0 only those days eligible after the statistical analyses de- An. To. 0.55 0.83 2.0 scribed above and included among the last 3 days on the Re. Da. 0.75 0.68 0.5 experimental diets were used in calculating excess urinary An. Na. 1.15 0.50 0.5 sulfur values. St. Po. 0.50 0.50 0.5 Cystathionine synthase deficient RESULTS Vi. Sw. 1.10 0.40 0.5 Ba. Re. 1.03 0.50 0.5 The homocystine excretion of cystathionine synthase- Fr. Mi. 0.58 0.30 0.5 deficient patients is known to be affected by two vita- Wi. Cr. 1.374 1.65$ 2.0 mins, pyridoxine (16, 17, 19) and folic acid (32, 33). Jo. Ho. 0.951 0.574 0.5 Ro. Kr. 0.98 0.40 0.5 Some patients respond to large doses of B6 with de- Ca. Me. 0.88 0.35 0.5 creases in plasma and urinary homocystine and methi- Cystathioninuric (presumptive y-cystathionase deficient) onine, whereas other, unrelated patients show no such Ge. Po. 1.20 0.35 0.5 response (16, 17, 19). These responses to B6 are thought to be genetically determined manifestations of hetero- * Supplied as either 2.0 mg in Dayalets-M tablet or as 0.5 mg in separate capsule. geneity in cystathionine synthase-deficient patients (18). t Value represents average of two measurements. Therefore, each patient was classified with respect to his pyridoxine responsiveness (Table I), and the ex- during the experimental periods than during the pre- periments were designed so that each patient would be ceding equilibration periods. in his basal state during the present studies, rather than Cystathionine synthase-deficient subjects tend to de- in pyridoxine-induced response. To determine relative velop abnormally low serum folate concentrations when pyridoxine intakes, urinary 4-pyridoxic acid excretions receiving normal folate intakes (32). To ensure that were measured. The excretion of this compound pro- all subjects were replete with folate during the present vides an index of Bs intake, being equivalent to ap- experiments, each was routinely given supplemental proximately 50% of the vitamin ingested by subjects on folic acid (2 mg twice weekly). Several patients had normal diets or receiving small supplements (34). For subnormal serum folic acid concentrations upon ad- the present studies, 4-pyridoxic acid excretions were mission to hospital (lowest, 3 ng/ml; normal, 5-21 measured during the latter half of both the equilibration ng/ml), but repeat determinations before, or during, the and experimental diet periods (Table II). During the experimental diet periods were in all cases within or equilibration periods no vitamin supplements were given, above the normal limits. and 4-pyridoxic acid excretions were close to or within the ranges reported for humans on normal diets by Nitrogen balance, excess urinary sulfur, and other workers using the same method of determination sulfur balance (0.27-1.08 mg/day [34]; 0.65-1.32 mg/day [28]). Dur- ing the experimental dietary periods the first five sub- Normal controls. A typical balance study for a nor- jects to be studied (Wi. Cr., Ba. Ha., Ba. Re., Ka. Ro., mal subject is shown in Fig. 1. At the outset of this and An. To.) received 2 mg pyridoxine daily in com- experiment the subject was changed from an equilibra- mercial multivitamin tablets and had slightly higher tion diet to an isonitrogenous experimental diet on urinary excretions of 4-pyridoxic acid than during the which the total methionine intake was 685 mg and the preceding equilibration periods when no vitamin sup- total cystine intake 10 mg. After the first 2 days, be- plement was given. Under no circumstances, however, cause of an apparent weight gain, the calorie intake did urinary 4-pyridoxic acid excretion exceed 1.65 mg, was reduced from 49.0 to 45.1 cal/kg, and the weight a value only slightly above the normal range. In subse- was effectively stabilized. On day 9 the methionine in- quent studies the pyridoxine HCl supplement was de- take was increased to 1,155 mg by the addition of fur- creased from the 2.0 mg supplied by the commercial ther supplemental free amino acid. The urine collections multivitamin tablets to 0.5 mg. On this regimen all for days 9 and 14 were known to be incomplete. The subjects had lower urinary 4-pyridoxic acid excretions creatinine excretion for day 7 indicated this collection

N Balance and S Excretion in Cystathionine Synthase Deficiency 1037 TABLE III Apparent Nitrogen Balance and Excess Urinary Sulfur: Normal Volunteers on Cystine-Poor Diet*

Sulfur amino acid intake Excess urinary Apparent nitrogen Subjecti Met Cys balance Nitrogen Sulfur mg/day g/day4(tSE) g/day ( :tSE) mg-atom/day ( 4SE) Females Ba. Ha. 662 9 +0.31±0.30 +1.3240.24 -0.3-0.1 St. Po.§ 661 3 +0.01±0.22 +0.72±40.22 -1.7±0.2 Ka. Ro. 670 11 -0.15±0.27 +0.54±0.22 -1.6±0.6 An. To. 659 10 +1.3240.49 +1.64±0.49 -1.1±1.3 Males Re. Da. 683 11 +0.22±0.38 +1.43±0.42 -1.6±0.4 Fr. Li. 667 8 -0.53±0.13 +0.36±0.13 -2.1±0.1 1,137 8 +0.39±0.36 +1.03±0.40 +0.1 +0.2 An. Na. 670 9 +2.30±0.45 +2.71±t0.44 -0.1±0.5 Jo. Ro. 685 10 +0.34±0.45 +1.16±0.40 -2.6±0.4 1,155 10 -0.43±0.24 +0.24±0.22 -1.8±0.5 * Experimental plan, diet, and methods of treating the data are described in Methods. The actual data from which these balances were calculated have been deposited with the National Auxiliary Publications Service (NAPS document no. 02536; ASIS/NAPS, Microfiche Publications, New York). The nitrogen intakes ranged from 10.49-11.07 g/day. Caloric intakes varied from 39.0-54.8 cal/kg body wt and for each subject had been adjusted before the experimental period and were then kept constant, so that during these periods weight changes were small. Ba. Ha. was exceptional in that the initial caloric intake was excessive and was decreased on day 3 of the study. During the study this subject gained 1.4 kg. I All subjects were normal volunteers between 18 and 24 yr old. § The equilibration diet for this subject was unusual and provided approximately 450 mg of methionine and 375 mg of cystine, with additional nitrogen furnished by supplemental L-glycine, bringing the total dietary nitrogen to approximately 10.9 g. to be an outlier (31). These days were eliminated from atom. The excess urinary sulfur decreased somewhat to consideration. It is seen that on these virtually cystine- -1.8±0.5 mg-atom for the last 3 days of this period free dietary regimens this normal control had mean included in the analysis. nitrogen excretions which deviated from absolute bal- In Table III is presented a summary of all similar ance by less than 5% of the total intake of 11.0 g of experiments on normal volunteers. Each of the four nitrogen. That is, the balances fell within what has male and four female subjects was either within the been defined by other workers as the "zone of nitrogen zone of equilibrium or in positive nitrogen balance on equilibrium" (35, 36). dietary regimens containing 659-685 mg methionine The total daily sulfur intake on the equilibration diet and 3-11 mg cystine. Urinary nitrogen excretions gen- for this experiment was 19.4 mg-atom sulfur, and the erally reflect the metabolic system and are not compli- daily urinary excretion was 18.7±0.6 (not shown in cated by the difficulties inherent in obtaining accurate Fig. 1). The intake was decreased to 4.67 mg-atom estimates of fecal nitrogen output during short-term sulfur on the experimental diet, whereupon the urinary metabolic balance periods. Therefore, data on the "ex- sulfur excretion decreased for 2 days, then remained cess urinary nitrogen," (calculated as total dietary ni- relatively constant. The excess urinary sulfur for this trogen - urinary nitrogen) are also compiled in Table experimental period was calculated using the values for III. These subjects excreted from 0.36 to 2.71 g less days 6 and 8, day 7 being eliminated as a possible out- nitrogen in their daily urine than their daily intakes on lier. For these days the mean urinary sulfur excretion diets containing 659-685 mg methionine and 3-11 mg was 7.25 mg-atom/day. Excess urinary sulfur thus was cystine. On the same regimens, the urinary sulfur losses equal to 4.67 - 7.25 = -2.6 mg-atom/day. Note that of most subjects slightly exceeded their dietary intakes, when urinary sulfur exceeds sulfur intake, the excess the maximum loss being -2.6 mg-atom sulfur/day. In- urinary sulfur has been assigned a negative value, in- creasing the methionine intake decreased (Jo. Ro.) or dicative of the fact that total bodily sulfur has decreased entirely ablated (Fr. Li.) the excess urinary sulfur loss. by at least this amount. The increase in the methionine To gain a preliminary indication of the relationship supplement on day 9 raised the total intake to 7.82 mg- between excess urinary sulfur and the sulfur balance 1038 J. R. Poole, S. H. Mudd, E. B. Conerly, and W. A. Edwards and how these parameters are affected by limitation of amino acids other than methionine and cystine, a fur- ID.1 S ther experiment was carried out with two normal con- a.-- trol subjects (De. Wa. and Ma. Sw.). After equilibra- tion periods, these subjects were given experimental diets analogous to the experimental diets used for the - -I.96 ±0i6--a0-038 0±27----- studies reported in Table III, except that the methionine ~~~~_iC intakes were increased to 1,209-1,219 mg in an effort to avoid excessive urinary sulfur losses. On this diet one subject (De. Wa.) was in nitrogen equilibrium (+0.30±0.13 g/day), whereas, unexpectedly, the second subject (Ma. Sw.) was below the zone of equilibrium but by less than 1 SEM (-0.65±0.11 g/day). Under these conditions, the excess urinary sulfurs were +0.8 and +0.1±0.6 mg-atom/day. Fecal sulfurs were also measured, and sulfur balances were calculated as total dietary sulfur - (urinary sulfur + fecal sulfur). Sulfur 63- losses in the stools were small relative to urinary losses,' and the resulting sulfur balances were +0.1 and -1.6-+- 62 0.5 mg-atom/day. 2 3 4 5 6 7 89e 2 314 JAYS --- I FIGURE 2 A study of a cystathionine synthase-deficient, ] B&-nonresponsive patient (Vi. Sw.) on daily cystine intakes of 155, 305, and 505 mg. Daily intakes of methionine and nitrogen were 655 mg and 10.36 g, respectively, throughout. Only 2 days of study could be completed on the intake of ______505 mg of cystine. See text and Fig. 1 for further ex- oh -+34 : _ planation. After 6 days on the experimental diet, the phenylala- nine supplement was decreased so that the subjects were .045 -0 43±1024 -- receiving only 100 mg daily of , a mar- ginal amount for maintenance of nitrogen equilibrium 71 on the tyrosine intake of these subjects (37). As ex- _~ pected, on this diet both subjects were near the lower end of the zone of nitrogen equilibrium (-0.51±0.13 --2.6 and -0.63±0.24 g/day). On the same regimen, sulfur ±0.4 ±O 5 losses increased so that the excess urinary sulfur values were -1.5±0.3 and -2.5±0.1 mg-atom/day, and the sulfur balances were -2.0±0.3 and -4.6+0.5 mg-atom/ day. Cystathionine synthase-deficient patients. A study of

7 a cystathionine synthase-deficient, Be-nonresponsive pa- tient (Vi. Sw.) is shown in Fig. 2. This study was <72-3J ~- 45 x similar in design to those carried out with the control 4 C1v,9 ; Kge subjects, except that each of the experimental dietary

..4 7 3 2 3 .4 regimens contained some supplemental cystine. The DAYS total daily intake of this amino acid was increased pro- FIGURE 1 A representative study of a normal volunteer gressively during the three periods from 155 to 305, subject (Jo. Ro.). Daily methionine intake s were 685 and 1,155 mg. Daily intakes of cystine and niitrogen were 10 'This finding was typical. The fecal sulfur content of 21 mg and 11.0 g, respectively, throughout. The figures for stool samples from normal control subjects and cystathionine apparent nitrogen balance and excess urinaLry sulfur excre- synthase-deficient patients on equilibration or sulfur parti- tions are indicated as mean+ 1 SE in g nitrogen/day and tion diets averaged 13.2±1.3% of the total urinary sulfur mg-atom sulfur/day, respectively. See text for further ex- (range 3.4-30.0%o). The samples from the patients did not planation. differ significantly from those for normal controls. N Balance and S Excretion in Cystathionine Synthase Deficiency 1039 duced to acceptable levels by increased cystine intake, but the nitrogen balance remained equivocally below the zone of equilibrium. During a second balance study .~I.-' (Table IV, study III), similar observations were re- corded with respect to urinary sulfur loss, but the nitro- gen balances remained low and the urinary nitrogen losses appeared to be inconsistent with the results of study II. During these studies the two periods of lowest 1" ._' " V- 1 .i cystine intakes had to be discontinued during the 5th - .1 ,f; V" and 4th days, respectively, because of nausea, irritability, 'i -.1 .. lassitude, and one episode of vomiting. These symptoms have been noted in subjects deprived of essential amino acids (7). Ba. Re. tolerated well the diets containing - 7 more cystine. Thus, the clinical results, as well as most of the chemical measurements, suggest that this patient has a cyst(e)ine requirement, but the present studies do not conclusively demonstrate this requirement be- FIGURE 3 A study of a cystathionine synthase-deficient, Be- responsive patient (Jo. Ho.) on daily cystine intakes of 312, cause of the failure of high cystine intakes to bring all 112, and 12 mg. Daily intakes of methionine and nitrogen parameters unequivocally within the control ranges. The were 677 mg and 10.64 g, respectively, throughout. The clinical status of Ba. Re. was such that she required urinary creatinine excretion for day 4 indicated this collec- multiple medications during these studies, and it is tion to be a statistical outlier. See text and Fig. 1 for further explanation. possible that one or more of these medications may have affected the results obtained. For the additional five cysthathionine synthase-defi- then to 505 mg. On the lowest cystine intake the nitro- cient patients studied (Table IV, and Fig. 3) the results gen balance of this patient was -0.96±0.15 g/day, a were quite different. For none of these patients was the value significantly below the zone of equilibrium. The nitrogen balance significantly below the zone of nitrogen value for excess urinary nitrogen was less positive than equilibrium, even on diets as low in exogenous cystine the value for any control subject (Table III). The content as could be experimentally attained. The urinary excess urinary sulfur for this period was -4.8±0.3 nitrogen losses for four of the five were within the con- mg-atom/day, a much more negative value than was trol range. observed for any control subject, even on lower cystine A parallel conclusion may be drawn from the results intakes (Table III). When the cystine intake was in- on sulfur excretion. On the lowest cystine intakes, the creased to 305 mg, the nitrogen balance for patient Vi. excess urinary sulfur excretions of these five patients Sw. was in the lower region of the zone of equilibrium. ranged from +1.1 to -2.3 mg-atom/day. Thus, under The excess urinary nitrogen remained somewhat low. these conditions the excess urinary sulfurs of these The excess urinary sulfur decreased to -3.8+0.3 mg- patients were less than those of Vi. Sw. or Ba. Re. but atom/day, a value still outside the range observed in similar to the excess urinary sulfurs of most control control subjects. The increase in cystine intake to 505 subjects on similar dietary regimens. Measurements of mg apparently decreased the excessive losses of nitrogen fecal sulfur were performed during the studies of Ro. Kr. and sulfur, although only 2 days could be completed and Ca. Me. The calculated sulfur balances were -4.9 on this dietary regimen. Thus, as expected on the basis ±0.2 and -3.4+0.1 mg-atom sulfur/day, respectively. of the results of Brenton et al. (9), this cystathionine 'Y-Cystathionase-deficient patient. Included in Table synthase-deficient, Be-nonresponsive patient failed to re- IV are the results of a study of Ge. Po., a patient with main in balance unless supplied with a substantial a presumptive block at another step in the transsulfur- amount of dietary cystine. ation pathway, the y-cystathionase reaction (21). On a The results obtained in two studies of a second non- diet virtually free of cystine this cystathioninuric, Be-re- responsive subject, Ba. Re. were less clear-cut. In the initial balance study (Table IV, study II) this patient Being regarded as "inert," this ingredient is not specified in was below the zone of nitrogen equilibrium and lost descriptions of the medications. Therefore, all medications taken by any patient during the course of this work were excessive urinary nitrogen and sulfur on the lowest analyzed for inorganic sulfate and total sulfur. The major cystine intake (109 mg).' The urinary losses were re- resulting uncertainty occurred in the case of Ba. Re., who received as much as 1.5 mg-atom inorganic sulfate and 0.2 2After these dietary studies had been completed, we be- mg-atom organic sulfur in her medications. It is difficult came aware of the not uncommon practice in the formula- to correct for these intakes because the portions absorbed tion of medications of adding calcium sulfate as an excipient. are not known (see footnote XT of Table IV). 1040 J. R. Poole, S. H. Mudd, E. B. Conerly, and W. A. Edwards TABLE IV Apparent Nitrogen Balance and Excess Urinary Sulfur: Cystathionine Synthase-Deficient or Cystathioninuric Patients on Cystine-Poor Diet with Varying Supplements*

Sulfur amino acid intake Excess urinary Apparent nitrogen Subjectsl Met Cys balance Nitrogen Sulfur mg/day g/day ( ±SE) giday ( ±SE) mg-atom/day ( ASE) Cystathionine synthase deficient Vi. Sw. 655 155 -0.9640.16 0.0040.17 -4.8±t0.3 655 305 -0.37i0.27 +0.04±i0.30 -3.840.3 655 505 +0.01±0.06§ +0.52±+0.06 Ba. Re. (study II) 667 109 -0.88±0.7711 +0.02±0.64 -5.9 to -7.611¶ 667 309 -0.22±0.51 +0.56i0.50 -3.8 to -4.0±0.8¶ 667 509 -0.67±+0.30 +0.45±0.86 -0.1 to -0.341.51 (study III) 662 8 -0.68±0.4911 +0.58±0.49 -5.7 to -7.3111¶ 662 1,008 -0.95±0.30 -0.10±0.34 +1.1 to -0.4i1.0¶ Fr. Mi. 822** 5 +1.0740.14 +1.79±0.15 +1.1±0.2 822 80 +2.16±0.33 +2.76±0.35 +1.9±0.3 822 155 +0.9940.41 +1.8240.43 -0.6±0.6 Wi. Cr. 682 7 +0.58±0.21 +1.48±t0.19 -2.3±0.3 682 107 +2.21±0.29 +3.1340.29 -1.2i0.3tt Jo. Ho. 677 12 +0.13±0.07 +0.75±0.05 -2.2±0.5 677 112 -0.43 40.22 +0.34±0.22 -0.5±-0.2 677 312 -0.34±0.13 +0.39±0.19 -0.5±0.3 Ro. Kr. 659 3 -0.6040.14 +0.14±0.13 -2.3±t0.2 Ca. Me. 644 4 +0.87±i0.19 +1.49±40.14 -1.3±0.1 I Cystathioninuric (presumptive -y-cystathionase deficient) Ge. Po. 673 6 -0.11±0.37 +0.71±0.38 -3.840.0 * See Table II and Methods. The actual data from which these balances were calculated have been deposited with the National Auxiliary Publication Service (NAPS document no. 02536). Nitrogen intakes ranged from 10.36 to 11.20 g/day. Caloric intakes varied from 36.5 to 59.5 calories/kg body wt and were adjusted as described in Table III. The initial intake for Wi. Cr. was excessive and during the period on 107 mg cystine this subject gained 1.9 kg. I See Table I for further details on these subjects. § Based on 2 days only, after which study was discontinued because of a death in the patient's family. 11 The studies of Ba. Re. on 8 mg and 109 mg cystine had to be discontinued during days 4 and 5, respectively, because of nausea, vomiting, and weakness. The excess urinary sulfur values are those for the last complete days of these studies. ¶ This patient was receiving some sulfur in her medications. The values for excess urinary sulfur were calculated on the alternative assumptions that (a) all of this sulfur was absorbed (yielding the less negative values for each study) or (b) none of this sulfur was absorbed (more negative values). ** An additional 170 mg of L-methionine was added to the diet to meet the requirement for this 15 yr-old boy (24). t This study was the initial patient study, and the experimental protocol differed slightly from the standard one adopted subsequently and described in Methods. This patient was gaining significant weight for the first 4 days on the experimental diet. His weight was stabilized after caloric adjustments on days 5 and 7. The excessive caloric intake may have accounted for the relatively high nitrogen retention during the dietary period he was receiving 107 mg cystine. 11 For this patient, in contrast to others studied, the daily urinary sulfur excretion did not reach a constant value until the 5th day on the experimental diet. Therefore, the mean excess urinary sulfur excretions averaged over the last 4, 3, and 2 days decreased progressively, being equal to -3.6± 1.5, -2.240.9, and -1.3 ±0.1 mg-atom sulfur, respectively. The last value is regarded as the most valid, and has been entered in the table. sponsive patient remained within the zone of nitrogen cess urinary sulfur was -3.8 mg-atom/day, slightly equilibrium with a balance of -0.11±0.37 g/day (Fig. more negative than the values observed for normal con- 4). His urinary nitrogen loss was acceptable. His ex- trol subjects (Table III). N Balance and S Excretion in Cystathionine Synthase Deficiency 1041 oninuric Ge. also increased Ge Po patient, Po., his plasma cyst(e)ine concentration in the face of severe cystine restriction. rl; L...

.. -. -t " 3 Urinary sulfur partition studies The partition of total urinary sulfur between sulfate and nonsulfate sulfur should provide an index of the . extent to which organic sulfur ingested as either -20. methionine or cystine has been metabolized to the com- -20 mon end product sulfate. Accordingly, urinary sulfur - -3.8 ±+3C partition studies were performed on five normal volun- teers, three cystathionine synthase-deficient, pyridoxine -2 0 responders, and two cystathionine synthase-deficient, -40 pyridoxine nonresponders who did not have residual -6.0 cystathionine synthase activities in their fibroblast ex- tracts Each received a constant natural -80 (18). subject diet calculated to provide half the dietary sulfur as -_Io 0 methionine and half as cystine. Urinary inorganic sul- 76 I-& fate, ethereal sulfate, and total sulfur were measured 75 daily. Nonsulfate sulfur was then calculated as total 74 L 50C)ca9/ eg sulfur minus total sulfate. In most cases values were f 52 0 c kg obtained with the subjects receiving only normal dietary 2 4 5 6 7 intakes of B8 and also on high doses of pyridoxine 2 3 4 5 6 7 83 (500 1-11 lAY S mg orally daily) (Table VI). In their basal states (i.e., while receiving no supple- FIGuRE 4 A study of a cystathioninuric, B-responsive pa- mental pyridoxine) normal control subjects excreted tient (Ge. Po.) on a daily cystine intake of 6 mg. Daily 8.-10.6% of their intakes of methionine and nitrogen were 673 mg and 10.86 g, total urinary sulfur as nonsulfate respectively, throughout. See text and Fig. 1 for further sulfur. The pyridoxine-responsive patients excreted explanation. 15.3-25.9% as nonsulfate sulfur, values that in each case were significantly higher than the highest value Plasma and urine amino acids found for a control subject (P < 0.01 for Jo. Ho. and Ja. Is.; P <0.05 for Th. Kr.). The percent nonsulfate Plasma and urine amino acid analyses were per- sulfur excretions of the non-Be-responsive patients (27.5 formed at least once during the latter half of each and 31.7%) were elevated above the control values but, dietary balance period. In the normal controls, no surprisingly, were not significantly higher than the changes were noted in either methionine or cystine corresponding values found for two of the responders levels, all being within normal limits on both the (Jo. Ho. and Ja. Is.). Among the responders, only equilibration and the experimental diets (data not Th. Kr. excreted significantly less nonsulfate sulfur shown). The values for patients are shown in Table V. than did the nonresponsive patients, 01. Ma. and Ba. Re. The most notable observations were the following: Pyridoxine administration did not cause a statistically After the change from the equilibration diets to the significant change in the percent nonsulfate sulfur ex- experimental diets which contained less methionine, the cretion of any normal control. On the other hand, each concentrations of methionine and homocystine in plasma of the three responsive patients showed a significant and the urinary excretions of homocystine decreased in decrease in nonsulfate sulfur excretion during pyridox- cystathionine synthase-deficient, Be-responsive patients ine treatment (P < 0.01). Under these conditions, the Wi. Cr., Ro. Kr., and Ca. Me. but not in responder percent nonsulfate sulfur excreted by responders Jo. Ho. Jo. Ho. Similar changes did not occur in nonresponders and Th. Kr. fell to values not significantly higher than Vi. Sw. and Ba. Re. Fr. Mi. was atypical among the the highest control value. The nonsulfate sulfur excre- Be-nonresponsive patients and displayed changes similar tion of responder Ja. Is. remained marginally elevated to those found among the responders. All responsive (0.05 < P < 0.10). For the nonresponders, pyridoxine patients maintained or increased their plasma cyst(e)ine therapy did not bring about a significant decrease in concentrations, even on the lowest cystine intakes. nonsulfate sulfur excretion. As a result, while the pa- Fr. Mi. manifested a similar tendency, although no tients were on pyridoxine, the percent nonsulfate sulfur plasma cystine was detected in one sample taken during excretion of each nonresponsive patient was significantly a period of relatively low cystine intake. The cystathi- higher than the corresponding value for each responder. 1042 J. R. Poole, S. H. Mudd, E. B. Conerly, and W. A. Edwards TABLE V Plasma and Urinary Amino Acids during Equilibration and Experimental Diets

Dietary intake Plasma amino acidst Urinary amino acidst Subject Diet* Met Cys Met j Cystine Homocystine Met Homocystine mg pum/mi pm/day Cystathionine synthase deficient Vi. Sw. Equilibration 1,451 924 354 368 Experimental 655 155 299 520 Experimental 655 305 340 932 Experimentall 655 505 355 1,118 Ba. Re. Equilibration 1,613 1,021 0.462 ND 0.120 194 429 Experimental 667 509 0.412 <0.015 0.118 155 348 Experimental 667 309 0.616 ND 0.122 216 432 Experimental 667 109 227 421 Equilibration 1,559 990 0.888 0.034 0.094 507 496 Experimental 663 8 Experimental 663 1,008 0.748 ND 0.069 88 147 Fr. M i. Equilibration 1,473 1,124 0.140 ND 0.148 204 297 Experimental 822 155 0.064 0.106 0.010 126 25 Experimental 822 80 0.070 ND 0.014 57 25 Experimental 822 5 0.070 0.098 0.012 94 50 Wi. Cr. Equilibration 1,511 912 0.400 0.034 0.062 185 220 Experimental 682 107 0.076 0.112 0.008 65 25 Experimental 682 7 0.070 0.066 0.007 108 41 Jo. Ho. Equilibration 1,469 1,005 0.098 0.028 0.056 67 333 Experimental 663 312 0.104 0.070 0.024 83 165 Experimental 663 112 0.146 0.090 0.046 49 254 Experimental 663 12 0.112 0.056 0.036 54 248 Ro. Kr. Equilibration 1,444 995 0.064 0.112 0.010 91 106 Experimental 659 3 0.050 0.126 ND 49 14 Ca. Me. Equilibration 1,505 984 0.608 ND 0.070 303 443 Experimental 644 4 0.090 0.042 0.028 91 156 Cystathioninuric (presumptive Ty-cystathionase deficient) Cystathionine Cystathionine Ge. Po. Equilibration 1,451 965 0.028 ND trace 104 585 Experimental 677 6 0.028 0.118 trace 83 370

* The equilibration diets were composed of natural foodstuffs isonitrogenous with the experimental diets which were semi- synthetic as described in Methods. The results are listed in the sequence that the studies were actually carried out. Sample from near the end of specified dietary regimen. ND, not detected; dash (-), sample not available. § Patient refused venipuncture. This experimental period lasted 2 days only (see Table IV).

DISCUSSION quired to achieve nitrogen equilibrium was then deter- mined. were The chief aim of the present work was to evaluate as The experiments designed to eliminate vir- quantitatively as possible the capacities of various pa- tually all cystine from the basal experimental diet, a tients with defects in the transsulfuration pathway to consideration that dictated the use of a semisynthetic biosynthesize cysteine from methionine. To this end, diet in which most of the amino acid intake was pro- we measured nitrogen balances of patients and controls vided in the form of the pure compounds. The diet maintained on semisynthetic diets containing low-ade- chosen was modelled after that often used in studies quate amounts of methionine but virtually free of of essential amino acid requirements (35, 38-40) and cyst (e) ine. The amount of supplemental cystine re- contained the essential amino acids plus arginine, histi-

N Balance and S Excretion in Cystathionine Synthase Deficiency 1043 TABLE VI dine, and tyrosine in the approximate amounts found in Total Urinary Sulfur and Percent Urinary Organic Sulfur 20 g whole egg protein. Supplemental glycine was added on Sulfur Partition Diet: Effect of Pyridoxine* to keep the experimental diets isonitrogenous with the equilibration diets (35, 38-40). This level and pattern Total urinary sulfur Organic sulfur of amino acid intake satisfied an additional desideratum Pyridoxine supplement Pyridoxine supplement to keep the methionine intake reasonably low to avoid Subject None 500 mg None 500 mg insofar as possible the accumulation of what might be 1&g-atom/kg ( 4SE) Percent ( iSE) considered "toxic" concentrations of methionine metab- Normal volunteers olites in the patients with transsulfuration defects. As Ba. Ha. 299±10 - 8.4+1.2 has been pointed out (41) this diet satisfies the mini- Ja. Ho.$ 356±35 354±34 10.3i1.5 8.1±0.9 mum essential amino acid requirements proposed by Cr. Ki.t 328 ±35 341 ±38 10.6±+-1.7 8.4 ± 1.4 Rose and Wixom (42) and others (43, 44). However, Ka. Ro. 334±15 - 9.0±1.4 recent results suggest that this regimen may be margin- An. To. 294±-40 275±i 19 9.5±i 1.0 12.2 ± 1.3 ally inadequate for maintenance of true nitrogen bal- ance (41, 45, in Cystathionine synthase-deficient B6-responders§ 46). Although, practice, our normal subjects on the basal semisynthetic diet containing vir- Jo. Ho. 265±12 276±11 25.9±1.7 11.940.9 tually no cystine Ja. Is. 313±4 293±24 25.5±1.8 16.0±1.2 remained within the zone of nitrogen Th. Kr. 304±t32 294±35 15.3i1.3 8.4±0.9 equilibrium (Table III), these relatively short-term studies do not prove the complete, long-term nutritional Cystathionine synthase-deficient B6-nonresponders l adequacy of the diets employed. Longer studies, or 01. Ma. 304424 266±26 31.7 ±2.4 31.2±i0.9 adoption of "corrected" rather than "apparent" nitrogen Ba. Re.¶ 283±12 280i5 27.5±1.8 26.3i2.5 balances (47) might have indicated that the basal diet used * was inadequate. Each subject was given a constant diet estimated to contain An additional criterion for comparison of normals methionine and cystine so that each contributed 140 ,ug-atom and sulfur/kg body wt. patients is afforded by the measurements of excess t 20 yr-old male. urinary sulfur performed in the course of our studies. § Residual cystathionine synthase activity was detected in Again, it is noted that these measurements do not test fibroblast extracts from each of these patients (18). the long-term adequacy of any particular dietary regi- 11 No residual cystathionine synthase activity was detected men. For this purpose, true balances taking into account in fibroblast extracts of either of these patients (18). fecal and other losses would be needed. 1 Corrected for sulfur content of medications taken by this subject. The maximum uncertainty in organic sulfur due to In Table VII the results on our present patients, as these corrections is 1%. well as the patient studied by Brenton and coworkers

TABLE VI I Patients with Transsulfuration Defects: B6-Responsiveness, Presence of Detected Residual Enzyme Activity, and Presence of Excessive Loss of Nitrogen or Sulfur on Lowest Cystine Intake Studied*

Excessive loss of: Residual enzyme Patient Defective enzyme B6-responsive detected Nitrogen Sulfur Stuart M.t Cystathionine synthase No Unknown Yes Unknown Vi. Sw. Cystathionine synthase No No Yes Yes Ba. Re. Cystathionine synthase No No ? Yes Fr. Mi. Cystathionine synthase No Yes No No Wi. Cr. Cystathionine synthase Yes Yes No No Jo. Ho. Cystathionine synthase Yes Yes No No Ro. Kr. Cystathionine synthase Yes Yes ? No Ca. Me. Cystathionine synthase Yes Yes No No Ge. Po. -y-Cystathionase Yes Unknown No Slight * Data on B6-responsiveness and enzyme assays are recorded or referred to in Table I. The nitrogen and sulfur losses are those for the lowest cystine intake tested for each patient (Table IV). f Studied by Brenton et al. (9). This patient was subsequently shown to be unresponsive to pyridoxine (D. C. Cusworth, personal communication). 1044 J. R. Poole, S. H. Mudd, E. B. Conerly, and W. A. Edwards (9), are summarized. It has previously been shown mal control cystathionine synthase activity is sufficient that there is a strong correlation between the presence to obviate this requirement. or absence of detected residual cystathionine synthase How much cyst (e) ine must be generated endogenously activity in cystathionine synthase-deficient patients and to avoid a requirement for exogenous cyst(e)ine in a their clinical responsiveness or nonresponsiveness to BR cystathionine synthase-deficient patient without residual administration. Residual cystathionine synthase activi- activity of this enzyme? The nitrogen balance of the ties were not detected in fibroblasts of 11 of 13 B6-non- Be-nonresponder studied by Brenton et al. on a daily responsive patients (18 and our unpublished results), cystine intake of 85 mg was -1.12±0.23 g/day (esti- nor in liver of two of two nonresponders (48). Among mated from Fig. 13 of reference 9). Nitrogen balance the patients in Table VII, Vi. Sw. and Ba. Re. fall into was restored with a cystine intake of 1,085 mg. Inter- the category of Be-nonresponsive patients without de- mediate intakes of cystine were not studied. The nitro- tected residual cystathionine synthase activity. Stuart M. gen balance of our patient Vi. Sw. was -0.96±0.16 is B6-nonresponsive. Although he has not yet been cate- g/day on a daily cystine intake of 155 mg. Nitrogen gorized with respect to residual enzyme activity, the equilibrium was restored and excessive urinary nitrogen results described above make it statistically likely that loss was prevented by an intake of 505 mg. The urinary he lacks such activity. Each of these three patients sulfur loss of Vi. Sw. remained slightly excessive on evidently lost excessive nitrogen and/or sulfur when 305 mg cystine. These studies suggest a cystine require- placed on low cystine intakes. These losses were re- ment of between 305 and 505 mg for Vi. Sw. The results versed by increasing the cystine intakes (except, ques- with patient Ba. Re. are more difficult to interpret be- tionably, for the nitrogen loss of Ba. Re.). cause nitrogen equilibrium was not restored with cer- In contrast to the results with most B6-nonresponsive tainty even on a cystine intake as high as 1,008 mg. patients, sensitive assays did detect measurable residual Judging by the clinical responses of Ba. Re. to various cystathionine synthase activities (0.1-10% of the mean cystine intakes and by her urinary sulfur losses, it may control value) in liver and/or fibroblasts extrasts of be tentatively estimated that 309-509 mg of cystine was each of the 25 responsive patients we studied (18, 19, adequate for this patient. Also pertinent are studies of 49), as well as seven of seven Be-responders studied by Bu. Ha., another cystathionine synthase-deficient B6- others (48, 50, 51). Wi. Cr., Jo. Ho., Ro. Kr., and nonresponsive patient without significant detected cysta- Ca. Me. are in this category of Be-responsive cystathi- thionine synthase activity in fibroblast extracts (18). onine synthase-deficient patients with detected residual The results obtained with this 8-yr-old boy were not enzyme activity. None of these patients were signifi- included in Table IV because he could not be persuaded cantly below the zone of nitrogen equilibrium or lost to accept the semisynthetic experimental diet. Accord- more sulfur than did our control subjects on diets vir- ingly, a normal food diet was devised, calculated to pro- tually free of cystine. vide 577 mg of methionine, 307 mg of cystine, and 4.13 Fr. Mi. falls into the relatively infrequently encoun- g of nitrogen. Supplemental glycine was added to bring tered class of cystathionine synthase-deficient Be-non- the total nitrogen intake to 10.7 g. On this diet, the responders with detected residual enzyme activity (18). nitrogen balance was +1.98±0.46 g and excess urinary Such patients illustrate that the presence of residual sulfur was -1.4 mg-atom/day. All of these observa- cystathionine synthase activity is a necessary but not tions together suggest that 310-510 mg cystine is ade- sufficient condition for Be-responsiveness. Fr. Mi. had quate for patients with virtually no residual cystathionine no excessive loss of either nitrogen or sulfur, even when synthase activity detected by present methods. Because his cystine intake was lowered to 5 mg daily. of the small number of patients studied, it should be Thus, among the eight cystathionine synthase-defi- clear that at best these estimates are approximations of cient patients studied to date, the five with residual the amount of cystine needed to prevent excessive nitro- enzyme activities could not be shown to require more gen or sulfur losses under our conditions. cystine than do normal subjects. There is no indication If cystathionine synthase-deficient patients without that for these patients cystine is an essential amino detected residual enzyme required 310-510 mg cystine acid. In contrast, for the three patients without de- under these conditions, it follows that the patients with tected residual enzyme activities, the evidence clearly residual cystathionine synthase activities who remained suggests a requirement for exogenous cystine in two, in equilibrium on diets containing adequate methionine, and the clinical signs and sulfur losses of the third pa- but at most 12 mg cystine, were forming endogenously tient are suggestive of such a requirement. It appears at least 300-500 mg (i.e., 2.5-4.0 mmol) cysteine. The that the generalization that cystine is an essential amino sulfur for this cysteine must ultimately have derived acid for cystathionine synthase-deficient patients (2, from methionine, since there was no other major dietary 10-17) is not universally correct and that in all likeli- sulfur source. The values of 2.5-4.0 mmol (which are hood the presence of a residual few percent of the nor- minimum estimates since our experiments would not N Balance and S Excretion in Cystathionine Synthase Deficiency 1045 detect the formation of more cysteine that would be re- of Gaull, Sturman, and Schaffner (55) generally support quired to sustain equilibrium) are equivalent to 20- the hypothesis that Be treatment brings about increases in residual hepatic cystathionine synthase activities of respon- 50% of the normal dietary intake of methionine of 8-12 sive patients. The mean basal activity in four responsive pa- mmol. The hepatic cystathionine synthase activities of tients under the conditions employed was 6% of the mean our Bo-responsive patients in their basal states (i.e., on control value; the mean activity during response, 18%o. Bo intakes provided by normal diets only) were 1-2% Porter, Grishaver, and Jones (56) found that extracts of fibroblasts from a Be-responsive patient retained 8% resid- of the mean control value (19, 49). Thus, it appears ual capacity to form cystathionine from homocysteine and that a residual activity of 1-2% of this enzyme endows serine, whereas no residual capacity to form cysteine from the patient with the capacity to convert at least 20-50% Na2S and serine was detected. of the normal methionine intake to cysteine. Our work- ACKNOWLEDGMENTS ing hypothesis as to the mechanism of action of B. in responsive patients is that vitamin administration brings We wish to thank Miss Paula Parisius and Mr. Byron Baer for performing the micro-Kj eldahl analyses, Drs. Josette about an increase of 2-4-fold in the basal enzyme activ- Bianchine, Richard Erbe, Stephen Goodman, Neil Holtz- ity of such patients (18, 19). The present demonstration man, John Littlefield, Victor McKusick, Grant Morrow, that the basal residual enzyme activity is capable of III, Andrew Sass-Kortsak, C. Ronald Scott, and David metabolizing 20-50% of the normal methionine intake C. Siggers for allowing us to study their patients, Miss this Ernestina Bou and Miss Mary Casey for aid with the diets, is clearly compatible with and supports working Mrs. Libby Ely and Mrs. Barbara Latrobe for social work hypothesis. To be able to metabolize the normal methi- assistance, and Mr. Larry Kleinman for help in the prepara- onine intake without undue homocysteine accumulation tion of the amino acid mixtures. Mrs. Karen Pettigrew was it may be necessary to possess no more than a rela- of invaluable assistance in carrying out statistical analyses tively small portion of the mean control cystathionine of the data. Dr. B. William Uhlendorf kindly provided synthase activity. fibroblasts for enzyme analyses. 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1048 J. R. Poole, S. H. Mudd, E. B. Conerly, and W. A. Edwards